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A simple and efficient method for analysis of plant growth regulators: a new tool in the chest to combat recalcitrance in plant tissue culture

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Abstract

This report presents a simple, rapid and accessible validated method for quantification of eight major plant growth regulators (PGR): cytokinins (6-(γ,γ-dimethylallylamino)purine (2-iP), benzylaminopurine (BA) and zeatin), auxin (indole-3-acetic acid; IAA), jasmonic acid (JA), salicylic acid (SA), gibberellic acid (GA3) and abscisic acid (ABA) by liquid chromatography mass spectrometry. This method was tested in eight species including agricultural, ornamental and medicinal species: St. John’s wort, African violet, banana, American elm, tobacco, potato, sweet wormwood, and fennel. The method has good reproducibility and good sensitivity with %RSD (percent relative standard deviation) between 1 and 10% for all matrices and recovery values of 89 to 118% for all analytes. Method detection limits were 50.65 ng/g, 203.4 ng/g, 50.65, ng/g, 50.65 ng/g, 203.4 ng/g, 12.7 ng/g, 193 pg/g and 3.08 ng/g, for SA, IAA, zeatin, JA, GA3, ABA, 2-iP, and BA, respectively. Our results with a range of plant species show that this method represents a simple, low-cost method for analysis of PGRs, and may also serve as an useful starting point for the analysis of other related PGRs, as demonstrated by inclusion of the SA derivative, acetylsalicylic acid, and the JA derivatives: 12-oxo-phytodienoic acid and JA-isoleucine. The efficiency of this method will enable its incorporation into the plant tissue culture work flow and through characterization of endogenous PGR levels, will allow for improved method development for recalcitrant species facilitating fundamental and applied studies in plant morphogenesis, propagation and conservation.

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Abbreviations

ABA:

Abscisic acid

ASA:

Acetylsalicylic acid

BA:

6-Benzylaminopurine

2,4-D:

2,4-Dichlorophenoxyacetic acid

GA3:

Gibberellic acid

IAA:

Indole-3-acetic acid

2-iP:

6-(γ,γ-dimethylallylamino)purine

JA:

Jasmonic acid

JA-Ile:

Jasmonic acid isoleucine

MeOH:

Methanol

MS:

Mass spectrometry

OPDA:

12-Oxo-phytodienoic acid

PCIB:

p-Chlorophenoxyisobutyric acid

PGR:

Plant growth regulator

%RSD:

Percent relative standard deviation

SA:

Salicylic acid

SIR:

Single ion recording

SJW:

St. John’s wort

SLV:

Single lab validation

TIBA:

2,3,5-Triiodobenzoic acid

TDZ:

Thidiazuron

UPLC:

Ultra-performance liquid chromatography

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Acknowledgements

The authors gratefully acknowledge the financial support of this work by the National Sciences and Engineering Research Council (NSERC) of Canada [Grant Number 46741] and the Gosling Research Institute for Plant Preservation (GRIPP) [Grant Number 050294].

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Authors and Affiliations

  1. Department of Plant Agriculture, Gosling Research Institute for Plant Preservation, University of Guelph, 50 Stone Rd W, Guelph, ON, N1G 2W1, Canada

    Lauren A. E. Erland, Mukund R. Shukla, W. Broc Glover & Praveen K. Saxena

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  1. Lauren A. E. Erland
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  2. Mukund R. Shukla
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  3. W. Broc Glover
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  4. Praveen K. Saxena
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Contributions

LAEE participated in conception and design, data acquisition, analysis and interpretation, MRS participated in conception and design, WBG participated in conception and design and data analysis and PKS participated in conception and design and data interpretation. All authors participated in manuscript preparation and gave final approval of the manuscript.

Corresponding author

Correspondence to Praveen K. Saxena.

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The authors declare no conflict of interest.

Additional information

Communicated by Pamela J. Weathers.

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Erland, L.A.E., Shukla, M.R., Glover, W.B. et al. A simple and efficient method for analysis of plant growth regulators: a new tool in the chest to combat recalcitrance in plant tissue culture. Plant Cell Tiss Organ Cult 131, 459–470 (2017). https://doi.org/10.1007/s11240-017-1297-1

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